An evolutionary study of the sequence of opal suppressor tRNA genes and their flanking sequences (i.e., in human, rabbit, chicken and Xenopus genomes) has been completed. Previously, a chicken and a human opal suppressor tRNA gene were isolated and sequenced and this past year the sequences of the corresponding genes, which had been isolated from rabbit and Xenopus genomes, were determined. The human and rabbit genes are strictly conserved as are those of chicken and Xenopus; and the mammalian genes differ from the others by a single pyrimidine transition at position 11. The gene, therefore, has been highly conserved which provides strong evidence that the gene products have an important cellular function. In addition, two gene products which are known to form phosphoseryl-tRNA arise from the single opal suppressor tRNA gene. The fact that these tRNAs are phosphorylated on the serine moiety and read the nonsense codon UGA suggest that they have a specialized and unique function which lies outside our present concepts of tRNA utilization. The tRNA gene products are present in minor levels intracellularly compared to other seryl-tRNA isoacceptors. Both the 5' flanking region, which has been highly conserved between rabbit and human genomes and has short stretches of homology in all four species, and the 5' internal control region, which has two extra nucleotides compared to the corresponding segment in all known tRNA genes, may have a regulatory role in reducing the level of transcription of the opal suppressor tRNA genes. These possibilities are being investigated by replacing the 5' flanking region with that of another tRNA gene and by site-specific mutagenesis of the 5' internal control region. Oligonucleotides have been synthesized which have a single or double base change in the 5' internal control region and in the anticodon and are being used in mutagenesis studies to convert the 5' internal control region to a normal tRNA control region and to convert the opal suppressor tRNA gene to an ochre suppressor.